Isolation and characterization of substance P-containing dense core vesicles from rabbit optic nerve and termini

In neurons, neuropeptides and other synaptic components are transported dow n the axon to the synapse in vesicles using molecular motors of the kinesin family. In the synapse, these neuropeptides are found in dense cove vesicl es (DCVs), and, following calcium-mediated exocytosis, they interact with r eceptors on the target cell. We have developed a rapid, large-scale techniq ue for purifying peptide-containing DCVs from specific nuclei in the centra l nervous system. By using differential velocity gradient and equilibrium g radient centrifugation, neuropeptide-containing DCVs can be separated by si ze and density from optic nerve (ON) and its termini, the lateral geniculat e nuclei and the superior colliculi. Isolated DCVs contain neuropeptides (s ubstance P and brain-derived neurotrophic factor), synaptic vesicle (SV) me mbrane proteins (SV2, synaptotagmins, synaptophysin, Rab3 and synaptobrevin ), SV-associated proteins (alpha -synuclein), secretory markers for DCVs pr eviously isolated (secretogranin II), and beta -amyloid precursor protein. By using electron microscopic techniques, DCV were also visualized and show n to be immunoreactive for neuropeptides, neurotrophins, and SV membrane pr oteins. Because of the interesting group of physiological and potentially p athophysiological proteins associated with these vesicles; this isolation p rocedure, applicable to other CNS nuclei, should represent an important res earch tool. (C) 2000 Wiley-Liss, Inc.